Glucan gels

ABSTRACT

The present invention relates to a glucan having a weight average molar mass of 15,000 to 50,000 g/mol on a single chain basis and a weight average molar mass in aqueous solution on an aggregate basis of 4 to 20×10 5  g/mol and existing in gel form in aqueous solution at a concentration ≧1% at 25° C. and neutral pH and having a melting temperature (gel to sol) of 35 to 60° C. when the glucan is dissolved in water at a concentration of 2%, methods for the production thereof, medical uses thereof, physical supports having the glucan applied thereto or impregnated thereon and in vitro methods of proliferation of skin cells which comprise contacting a population of skin cells with the glucan.

This application is a filing under 35 USC 371 of InternationalApplication No. PCT/GB2011/052358, filed 29 Nov. 2011, which claimspriority to GB Application No. 1020191.1, filed 29 Nov. 2010. Theseprior applications are incorporated herein by reference.

The present invention relates to a new glucan product, to processes forits manufacture and to uses thereof as a pharmaceutical, incorporated ina medical device, as a nutraceutical, cosmetic product or the like.

Glucans are a heterogeneous group of glucose polymers found in the cellwalls of plants, bacteria, fungi and protozoa. Glucans have a backbonechain and in some cases side chains which, depending of the origin ofthe glucan, comprise β(1,3), β(1,4) and/or β(1,6)-linked glucosyl units.Depending upon the source and method of isolation, beta-glucans havevarious degrees of branching and type of linkage in the backbone andside chains. The frequency and type of linkage in the side chains ishighly relevant to the molecule's biological activity. Glucans alsodiffer highly in their molecular weight as well as in their tendency forchain aggregation which both are essential features for the efficacyprofile of these molecules. Most beta-glucans of fungal and yeast originare in their native state insoluble in water, but can be made solubleeither by acid hydrolysis or by derivatization introducing foreigngroups like -phosphate, -sulphate, -amine, -carboxymethyl and so forthto the molecule.

In Europe, Asia and USA, beta-glucans especially from Bakers' yeast havelong been employed as feed additives for animals, in cosmetics, asdietary supplement for humans, as immunomodulators e.g. in treatment ofwounds, and as an active ingredient in skin cream formulations. Glucanshave been employed in the treatment of cancer as shown in WO02/058711.Beta-glucans are, in this context, regarded as immunostimulantsincreasing the activity of white blood cells partly by inducing wellregulated and site restricted inflammatory reaction localised to thecancer. Their use in the treatment of inflammatory bowel disease hasalso been described in WO 2009/063221. Further applications of glucanswithin wound treatment are described in EP 815144 and in U.S. Pat. No.6,875,754 as well as for the treatment of asthma and allergy asdescribed in U.S. Ser. No. 12/528,215.

Cereal glucans comprise generally unbranched chains of 3(1,3) and asignificant share of β(1,4) linkages, while yeast glucans are made up ofpredominantly β(1,3) linked glucosyl residues with β(1,6) linkagesacting as branch points for side chains which may comprise both β(1,3)and β(1,6) linked glucosyl residues. Other molecules classed as glucansinclude curdlan, a basically linear molecule made up of β(1,3) linkedglucosyl residues without branches. Lentinan is a glucan with a β(1,3)linked backbone but incorporating single β(1,6) linked glucosyl residuesattached essentially regularly to the backbone giving a haircombstructure of this molecule. The single β(1,6) linked glucosyl residuesattached to the backbone equivalent to a β(1,3,6) linkage point but nofurther molecules are attached to this linkage point and thus glucanslike lentinan do not have side chains. Other examples of this group ofglucans are scleroglucan, laminarin and schizophyllan.

Variations in branching and the length and structure of the side chainslead to contrasting secondary and tertiary structures and thusbiological activities. The higher order structures of glucans varyconsiderably and molecular weight, solubility and particle size will allinfluence activity in a generally unpredictable manner. Some productsare extremely potent inducers of inflammatory cytokines in target cells,whereas others have the opposite effect, completely inhibiting cytokinerelease. Typical for many insoluble beta-glucan products is theinduction of a whole range of inflammatory responses, where e.g.injection of insoluble beta-glucan formulations has been associated withgranuloma formation, arthritis induction and increased susceptibilityagainst gram negative sepsis. On the other side, soluble beta-glucansare not reported to be encumbered with such negative side effects, buttheir efficacy as immunostimulants have been known to varysubstantially.

It has been shown (WO 95/30022), for example, that a glucan productderived from yeast which has been modified by glucanase treatment toselectively remove (1,6) linked side chains is more potent instimulating the immune system of fish than a product with intact (1,6)linked side chains.

Glucans have great potential as therapeutic agents and adjuvants but thevast range of structural variability, problems of analysis with suchlarge and complex molecules and the lack of understanding aboutmechanism of action and receptors for these molecules, means that thereis still a great need for an improved glucan product with tailor-madebiological activities, and for controllable and repeatable processes formanufacture of homogeneous products.

Beta-glucans are known to be so-called Pathogen Associated MolecularPatterns as they are found at the surface of a number of pathogenic(micro)organisms, especially fungi. Higher organisms have thus evolvedmechanisms for recognizing these types of structures in order to findand destroy intruders belonging to this class of organism. In mammalsthe so called innate immune cells express specific receptors recognizingbeta-glucans, and one of the most prominent receptors is calledDectin-1.Other receptors are also involved in the recognition or signaltransduction induced by beta-glucans, amongst these are CD11b/CD18(CR3), and toll receptors 2 and 4 (TLR2 and TLR4). Of the cells involvedin recognizing beta-glucans are the typical phagocytes of the innateimmune system , i.e. monocyte, macrophages, dendritic cells, andgranulocytes, but also Natural Killer cells as well as a number ofendothelial cells and other more tissue specific cells have the abilityto express beta-glucan receptors.

The crucial step in inducing a biological response in the target cellsis the initial binding to the receptor and furthermore, it seems, theability of the beta-glucan formulation to cross-link a sufficient numberof receptors in order to induce an adequate signal-transduction into thecell. The present invention describes a product and a method for makinga product that has the ability to cross-bind receptors inducing aspecific type of biological activity. This is in contrast to insolubleproducts that could induce a massive response by cross-binding a largenumber of receptors and secondly be phagocytosed, which due to thenature of the insoluble (or “crystalline like”) glucan leads tolysosomal rupture within the cell inducing NLRP inflammasome activation.Insoluble beta-glucans may also induce

ROS (reactive oxygen species) that also would trigger inflammasomeactivation leading to an unfavorable inflammatory reaction. The currentinvention describes beta-glucans products that are able to induce asignificant inflammatory response that would activate several immunemechanisms, but without triggering inflammasome activation that istypical for a number of (aggregated insoluble) beta-glucan products.

The present invention potentiates glucan efficacy by establishing apharmaceutically beneficial supramolecular structure in the finalproduct.

The importance of higher order structure amongst β-glucans and thecontribution of the character of both individual glucan strands orchains and the higher order structure to the overall activity of theglucan product is described by Sletmoen et al. in Biopolymers vol. 89,No. 4 pp 310-321, 2008. Higher order structure may comprise a regulararrangement such as a triple helix or a more loose aggregation.

The present invention provides a glucan formulation that is perceived asa moderately sized entity when encountered by the target cells, but whenphagocytosed the glucan is easily taken up into phagosomes withoutinducing lysosomal rupture. The present invention thus describes a novelorganization of a highly potent soluble beta-glucan with good gellingproperties. Without wishing to be bound by theory it seems that theglucan molecules are arranged in a type of higher complex and loose“haystack” arrangement kept together by relatively weak hydrogen bondsbetween the frequent —OH groups along the glucan backbone structure. The“haystack” organization has the potential of presenting a number ofsites on its surface available for recognition by specific glucanreceptors on the target cells. The “haystack” organized molecules donot, however, harbor the rigidity of an insoluble product, but wouldmuch more easily become “degraded” and thus “immobilized” at the site orafter phagocytosis. Such a large higher order organization isadvantageous as compared both to insoluble and to known solubleproducts, since it gives an immunomodulatory response mimicking many ofthe effects observed with particulate and insoluble beta-glucans withoutinducing less controllable and possible harmful effects known to beassociated with insoluble beta-glucans.

In one aspect the present invention provides a glucan having a weightaverage molar mass on a single chain basis of 15,000 to 50,000 g/mol anda weight average molar mass in aqueous solution on an aggregate basis of4 to 20×10⁵ g/mol, said glucan existing in gel form when dissolved inwater at a concentration ≧1% at 25° C. and neutral pH and having amelting temperature (gel to sol) above 30° C., preferably between 35° C.and 80° C., more preferably between 35 and 60° C., still more preferablybetween 37° C. and 60° C., most preferably about 40° C. when the glucanis dissolved in water at a concentration of 2%.

Preferably the glucan is in aqueous solution at a concentration of 1.5to 6%, more preferably 1.5 to 5%, still more preferably 2 to 4%, mostpreferably about 2%. It is understood that a “gel” form can beconsidered an aqueous solution.

In a preferred aspect the glucan is a beta glucan, preferably it has abackbone of β(1,3) linked glucosyl residues and side chains of β(1,3)linked glucosyl residues (e.g. side chains of at least 2, 5, 10 or 20linked glucosyl residues) attached thereto via a β(1,6) linkage.

“Neutral pH” means pH 7.

A “single chain” refers to an individual glucan molecule, i.e. one inwhich the glycosyl residues are covalently linked. “Aggregates” formthrough hydrogen bond interactions and define a supramolecular or higherorder structure. Such associations are less permanent than provided bycovalent bonding but the methods described herein result in recognisablepatterns of aggregation, whose average molar mass can be analysed usingthe techniques referred to herein. The “aqueous solution” is typicallypH 7.

Alternatively viewed, the present invention provides a gel glucanproduct comprising glucan in aqueous solution at a concentration of 1 to6%, the glucan having a weight average molar mass on an aggregate basisof 4 to 20×10⁵ g/mol and a weight average molar mass on a single chainbasis of 15,000 to 50,000 g/mol, the gel glucan product having a meltingtemperature (gel to sol) above 30° C., preferably between 35° C. and 80°C., more preferably between 35 and 60° C., still more preferably between37° C. and 60° C., most preferably about 40° C.

Glucan products are usually particulate or in some cases soluble. Gelforms are very unusual, but the present gel product has been found toprovide excellent biological activity, in particular in wound healing,as compared to other glucan products. In wound healing it is of utmostimportance to apply a pharmaceutical or medical device in a manner whichsecures the moisturization of the wound and the products must cover andstick to the wound surface to avoid infections and provide for anadministration profile as deemed relevant by a medical practitioner ornecessary due to the type of wound. Usually, glucans in theirparticulate, semi-soluble or liquid form do not solve these basicrequirements either because they are not effective, they are in a statewhich is not applicable for wound healing purposes, or both. The glucanof the present invention combines these necessary characteristics thusmaking it useful for all applications where a pure glucan gel may find aproper use. In addition to strictly topical applications, other possibleuses could be oral and/or mucosal administration, such as treatingdiseases of the gastro-intestinal tract or the oral cavity in additionto cancer therapy for which the present gel product has been found tohave excellent activity. The excellent adhesion properties of the glucanaccording to the present invention enable it to cover the mucosal liningat the site of action and thus accelerate the healing process. Thus theglucans of the invention have particular utility in the treatment oforal mucositis and other indications affecting the mucosa.

According to the present invention a novel organization of a solubleglucan into a gel structure can be obtained by treating an adequatelyconcentrated solution of soluble glucan with an agent able to dissociatehydrogen bonds both between and within glucan chains, followed by addingan agent rapidly able to restore inter- and intra-chain hydrogen bondinginteractions. The supramolecular tertiary, or 3-dimensional, structureof a glucan, in this case the arrangement of the molecular chains withinthe glucan product as a whole, appears to be of utmost importance forefficacy. Without wishing to be bound by theory it seems that onlybiologically effective molecular structures provide for binding todifferent receptors at the target cells. Single chain, short chain orproducts not structured in an appropriate 3-dimensional complex mannerwill not be able to stimulate the body's immune system in the same way.

There are limited ways to characterize the 3-dimensional (also definedas tertiary or supramolecular structure) molecular structure of a gelcomprised by its single chains. General ways of describing such a gelcan be by the average molar mass and molar mass distribution of thesingle chains, as well as by physical characteristics such as viscosity.In the case of immunomodulating products, gels can also be indirectlydescribed by their biological efficacy profile, or in other wordsmeasuring of the so-called “biological fingerprint”. When usingmolecular mass as a defining physical characteristic, it is recognisedthat the analysis methods are generally destructive, leading to theanalysis of the single chain components of the gel product, or smalleraggregated structures, rather than giving a detailed picture of themolecular interactions between these single chains which are necessaryto give a biologically effective 3-dimensional supramolecular structure.Nevertheless a detailed analysis of several other physicalcharacteristics of glucans including their viscosity combined with abiological efficacy profile will enable the skilled man to distinguishbetween a variety of different glucans. One of these criteria is aspecific molecular mass range.

The molar mass of glucans can be determined in different ways. In thecase of a soluble glucan product the molar mass is conveniently measuredby SEC-MALS-RI (size exclusion chromatography with multi-angle lightscattering and refractive index detection) analysis, and such analysisprovides a weight average molar mass value (M_(W)) for the sample aswell as the distribution of different molecular weights within thesample. In the present invention, the weight average molecular mass(M_(W)) is defined as follows:

$M_{w} = {\frac{\sum{n_{i}M_{i}^{2}}}{\sum{n_{i}M_{i}}} = \frac{\sum{c_{i}M_{i}}}{\sum c_{i}}}$Where n_(i) is the number of molecules with molar mass M_(i). The weightconcentration c_(i) of molecules with molar mass M_(i) is proportionalto the molar mass M_(i) and the number of molecules n_(i).

c_(i) = M_(i)n_(i) =  > n_(i) = c_(i/M_(i))The weight concentration for each slice of the chromatogram is measuredby the RI-detector while the molar mass for each slice in thechromatogram is measured by the MALS-detector in combination with theRI-detector. The calculations are based on light scattering theory.

Specifically, the average molar mass (for single chains) according tothe present invention is determined by SEC-MALS-RI in DMAc with 0.5%LiCl (dimethylacetamide with 0.5% lithium chloride) assuming a do/dc of0.12 for the glucan in this solvent. The DMAc/LiCI solvent fullydissolves the said glucan into single chains, and subsequent SEC-MALS-RIanalysis with DMAc with 0.5% LiCI as eluent therefore gives a measure ofthe molecular weight distribution on a single chain level. In short, theanalysis of the glucan in DMAc/LiCI involves dissolution of the dryglucan in the solvent at a concentration of approximately 3 mg/ml bystirring the solution at r.t. over night and heating it at 100° C. for 1h, prior to the analysis by

SEC-MALS-RI using 3× PLgel Mixed-A LS columns and DMAc with 0.5% LiCI aseluent. The weight average molar mass for the glucan of the presentinvention on a single chain basis determined by this method is 15,000 to50,000 g/mol, preferably 25,000 to 45,000 g/mol, and more preferably30,000 to 40,000 g/mol.

In aqueous solution the weight average molar mass of the mainly higherorder structures and aggregates present is 4-20×10⁵g/mol, preferably5-15×10⁵ g/mol, and more preferably 6-12×10⁵g/mol. These averages arepreferably calculated when very large aggregates, i.e. molar mass above1.0×10⁷ g/mol, are excluded. The analysis of the glucan in aqueoussolution involves diluting the gel solution to approximately 3 mg/ml in0.1 M NaNO₃ with 0.02% NaN₃, heating to 100° C. in a capped glass tubefor 30 min, cooling to room temperature, filtrating through a 0.2 μmsyringe filter, and analysis by SEC-MALS-RI using TSKgel G5000PWXL+TSKgel G4000 PWXL columns and 0.1 M NaNO₃ with 0.02% NaN₃ aseluent. Similar set-ups with for example 0.05 M Na2SO4/ 0.01 M EDTA assolvent/eluent gives equivalent results. The combination of molar massvalues for the single chains and the higher order structures/aggregatesin aqueous solution gives a good indication of the molecular andsupramolecular structure of the gel as a whole and usefully defines theglucans of the present invention.

The glucans of the present invention are further characterized by beingin gel form at 25° C. and at a pH between 3 and 8. The glucan gels ofthe invention are further characterised by their viscosity profileexemplified by the melting temperature of the gels (gel to sol) of above30° C. and up to approximately 80° C., preferably above normal bodytemperature, more preferably between 37° C. and 60° C., most preferablybetween 39° C. and 60° C., e.g. 40-50° C. The figures above are givenfor a glucan gel in a concentration of 2% in an aqueous solution.

The gel melting point for a glucan product, i.e. the gel→sol transitiontemperature, is conveniently determined by small strain oscillatorymeasurements using a Stresstech HR rheometer or similar and examiningthe viscoelastic changes during cooling (70→10 ° C.) and heating (10→70° C.) of the glucan solution. An example of storage modulus (G′) plottedagainst temperature in such an experiment is shown in FIG. 1. Themelting temperature for this particular sample is equivalent to wherethe storage modulus of the curve for increasing temperature levels out(at approx. 0 Pa,), which is approx. 40° C. Another way of determiningapproximate melting temperature of the gel is to measure the viscosity(e.g. using a rotational viscometer) of the gel at sequentially highertemperature until the viscosity is essentially gone and the gel hastransformed into a solution. The gel melting temperature is preferably30-80° C., preferably over body temperature to guarantee a stabilizedglucan gel for topical applications. Topical administration demands acomparably lower melting temperature than oral administration oradministration to a site of an infection.

The glucan gel of the present invention is an aqueous gel and while thegel form can be confirmed by visual inspection, the viscosity and thepseudoplastic and thixotropic nature of the glucan gel may also bedetermined by viscosity measurement e.g. by using a rotationalviscometer. A 2% glucan gel according to the present invention has aviscosity of at least 1000 cP, preferably at least 1500 cP, measured at25° C. and a rotational speed of 10 rpm using a Brookfield DV-II+ProProgrammable viscometer with a small sample adapter and spindle SC4-31(corresponding to a shear rate of 3,40 sec⁻¹). A convenient method formeasuring the viscosity of this pseudoplastic and thixotropic gel is touse a so called up-down rate ramp, for example starting at 2 rpm andgoing up in 2 rpm increments to 10 rpm and then going back down again in2 rpm steps. The data from such an experiment can both demonstrate thepseudoplastic (decreasing viscosity with increasing shear rate) andthixotropic (decreasing viscosity over time while subjected to shear)characteristics of the gel as well as provide a measure of e.g. 10 rpmviscosity.

The glucans of the present invention are typically derived from yeast,preferably from Saccharomyces cerevisiae. The basic molecular structureof these glucans is typically a β-1,3-backbone (meaning a chain ofglucose molecules linked by β-1,3 linkages), in addition to β-1,3 sidechains (meaning a chain of at least two glucose molecules linked by β-1,3 linkages) and a β-1,3,6-linkage point linking the side chains to thebackbone. In addition, glucans from yeast comprise β-1,6 linkages whichmay be linked to the side chains or directly to the backbone. Furthertypes of linkages do exist but at a comparably low level. Other yeastswhich may provide a source for the glucan include Brewers yeast, Candidasp. like Candida albicans, Candida cloacae, Candida tropicalis, Candidautilis, Hansenula sp. like Hansenula wingei, Hansenula ami, Hansenulahenricii and Hansenula americana, Histoplasma sp., Kloeckera sp.,Kluyveromyces sp. like Kluyveromyces lactis,

Kluyveromyces fragilis, Kluyveromyces polysporus, Pichia sp.,Rhodotorula sp., Saccharomyces sp. like Saccharomyces delbruekii,Saccharomyces rosei, Saccharomyces microellipsodes, Saccharomycescarlsbergensis or different Saccharomyces strains like Saccharomycescerevisiae R4 (NRRL Y-15903) and R4 Ad (ATCC No. 74181), Schizophyllumsp., Schizosaccharomyces sp. like Schizosaccharomyces pombe, Torula sp.and Torulopsis sp.

However, the gel glucans of the present invention may be derived fromother suitable sources, e.g. bacterial, fungal or cereal glucans. Thetherapeutic activities of various glucans are well documented in the artand the processes of the present invention may be used to enhanceactivity of glucans in general, in particular in wound healing where thephysical form and inter-molecular structure of the glucan product hasbeen shown, by the present inventors, to be particularly significant.Without wishing to be bound by theory a rule of thumb is that the higherthe weight average molar mass on a single chain basis of the glucan usedaccording to the present invention, the more efficacious glucan gels maybe produced.

The side chains of the glucan gels of the present invention usuallycomprise 2 or more β(1,3) linked glucosyl units. According to thepresent invention, single molecules linked to a main chain are notregarded as “side chains”.

The glucans of the present invention preferably have side chains of,i.e. consisting or consisting essentially of, β(1,3) linked glucosylunits. In addition to the β(1,3) linked side chains, the glucans mayalso have one or more β(1,6) linked side chains. By altering the chainsof the structure it is possible to alter the characteristics of thefinal product. There are many different ways of altering glucansincluding enzyme-treatment, use of acids like formic acid orhydrochloric acid or different bases as well as by other means.Preferred glucans are those which have been treated by acid (e.g. formicacid) or enzyme or any other suitable method to significantly reduce oreliminate the number of repetitive (1,6)-linked glucose molecules withinthe glucan. These (1,6)-linked glucosyl moieties would normally be foundin the side chains of beta-glucans derived from yeast. The resultingglucans have β(1,3) main chains and β(1,3) side chains which are linkedthereto through a single β(1,6) linkage which is not cleaved off by theelimination treatment. The preferred glucans are essentially free ofrepetitive β(1,6) linked glucosyl residues. The single (1,6) linkages atthe branch points (the β(1,3,6)-branching points) do not provide‘repetitive’ β(1,6) linked glucosyl units. By ‘essentially free’ ismeant less than 6%, preferably less than 4% and most preferably lessthan 3% of the total glucosyl units.

Some treatments, such as enzyme treatments, may leave up to 4beta-1,6-linked, but typically 2 beta 1,6 linked glucosyl unitsuncleaved in the side chains. Such molecules are also ‘essentially free’of repetitive beta 1,6-linked glucosyl units.

The distribution of linkages within preferred glucans of the inventionmay be represented as follows:

Type of linked glucosyl residue % β(1, 3) 80-98 β(1, 6) 0-6 β(1, 3, 6)1-8 Terminal 0.01-6  β(1,3,6) refers to branch point residues which are (1,3) linked in thebackbone and participate in a (1,6) connection to provide a side chain.

The glucan of the present invention could be in the form of a single,extracted fraction or two or more different fractions with differentaverage molecular weights.

The glucans are underivatized in terms of chemical modifying groups.

The glucans of the invention are generated by a novel process. Theinventors have found that treating an adequately concentrated solutionof soluble glucan with an agent able to dissociate hydrogen bondsbetween glucan chains, followed by adding an agent able to restoreinterchain interactions, a novel gel glucan product is obtained withimproved activity as compared to other similar glucan products. By doingthis a highly randomly organized “haystack” gel will be created withouthaving the typical triple helical structure of “annealed” beta-glucanchains. Surprisingly it was observed that this type of gel-structure wassignificantly more potent as immunomodulator than a classical organizedsoluble beta-glucan either in triple helical conformation or multiplesof helixes.

Thus in a further aspect the present invention provides a method ofproducing a gel glucan product as defined above wherein an aqueoussolution of glucan molecules is treated with an agent in order todissociate hydrogen bonds between the glucan chains and then treatedwith an agent which enables the reformation of hydrogen bonds betweenthe glucan chains.

Thus, the present invention provides a method of producing a glucan gelcomprising the following steps:

a) treating an aqueous solution of glucan molecules with an agent ableto dissociate hydrogen bonds; and

b) adding an agent rapidly able to reform hydrogen bonds.

Alternatively viewed the present invention provides a method ofproducing a gel glucan product as defined herein, said method comprisingthe following steps:

a) treating an aqueous solution of glucan molecules with an agent whichdissociates the glucan's hydrogen bonds; and

b) contacting the product of step a) with an agent which enablesreformation of hydrogen bonds within the glucan.

The dissociated and reformed hydrogen bonds may be intramolecular, i.e.within a single chain or intermolecular, between chains resulting in theformation of aggregates.

Preferably the agent able to dissociate hydrogen bonds is an alkalisalt. Preferably the agent able to dissociate hydrogen bonds is used ata final concentration of above 50 mM, preferably about 150 mM.

Step a) is preferably performed at a temperature of 10° C. to 25° C.,more preferably 15° C. to 20° C., most preferably about 18° C.

The addition of the agent able to dissociate hydrogen bonds ispreferably performed slowly, preferably at a rate of approximately 1litre per minute of 24 moles of NaOH dissolved in 10 litres of water,being added to 200 litres of 2% glucan solution or at an equivalent rateif the volumes or concentrations are different.

One such agent to dissolve hydrogen bonds between OH-groups in thepoly-glucose chain would be sodium hydroxide (NaOH) in a sufficientconcentration that would deprotonise the numerous OH-groups in thechains. This would lead to a complete dissociation of all intermolecularbonds typical for these high molecular weight glucans resulting in arandom organization of the chains in solution. By neutralizing thesolution by addition of acid to neutralize the alkali, the OH-groups arereformed and new hydrogen bonds between the chains can be established.

Using NaOH as the agent would typically need the addition of e.g. 2MNaOH solution to a final concentration of above 50 mM, or morepreferably about 150 mM to a soluble glucan concentration of 1-6% inaqueous solution, more preferably 1,5-4% or most preferably 2-4%.

The step of reforming hydrogen bonds can also be viewed as neutralizingthe solution following the addition of the agent able to dissociatehydrogen bonds.

Since the agent able to dissociate hydrogen bonds is preferably analkali salt, then preferably the agent rapidly able to reform hydrogenbonds, i.e. the agent which neutralizes the solution produced in stepa), is an acid, preferably a strong acid. Preferably the agents in stepa) and step b) of the method are added in equimolar amounts. Forinstance, if 2M NaOH solution was added in step a) then in order toneutralize the solution an equimolar amount of e.g. 2M hydrochloric acid(HCl) can be added to the solution. Preferably the neutralization stepis performed under agitation for a brief period which is long enough toensure an efficient neutralization. For a volume of 1000 ml this stepcould be performed in less than 1 minute, e.g. less than 30 seconds. Asshown in the Examples, larger volumes would necessarily take longer forall the acid to be added and mixed. After this the solution is left toestablish the gel-conformation, for a volume of 1000 ml the time takenfor gel formation may be 1 to 10 minutes, longer for larger volumes.

A rapid reformation of hydrogen bonds can be assessed in terms of thespeed of gel formation. If a gel forms in less than 15 minutes fromfirst addition of the agent (which can be considered a renaturing agent)then this is indicative of rapid reformation of hydrogen bonds, although“rapid” will typically mean less than 10 minutes, preferably less than 8or 6 minutes, more preferably less than 4 minutes or 3 minutes. It beingnonetheless appreciated that the larger volumes will generallynecessitate longer time periods for gel formation/hydrogen bondreformation.

Any other agent having the ability to dissociate the hydrogen bondscould replace NaOH, and any other agent able to rapidly allowre-establishment of the hydrogen bonds forming a “haystack” type of gelcould replace HCl. The skilled man is aware of other agents which candisrupt and then restore hydrogen bonds, bases and acids areparticularly convenient as one can be readily balanced against the otherto neutralize the impact of the agent which has disrupted hydrogenbonds. Other strong acids such as formic acid or sulphuric acid may beused. Also other alkali salts including, but not limited to, potassiumhydroxide, lithium hydroxide, and calcium hydroxide, as well as possiblyso called superbases such as sodium hydride or sodium amide, can bepotential agents for deprotonation and disruption of hydrogen bonds. Anyacid with the appropriate quality can be utilized to neutralize thesolution in order to restore hydrogen bonds—this includes, but notlimited to, phosphoric acid, acetic acid, and citric acid. Urea orformamide are also commonly used to disrupt hydrogen bonds and couldpossibly be used in this process.

It will be appreciated that in a system involving large and complexorganic molecules, it is not feasible or necessary to ensure that allhydrogen bonds have been disrupted or that all molecular chainsparticipate in significant hydrogen bonding after conditions have beenapplied which enable the restoration of hydrogen bonding. However, theconditions applied will be such as to radically alter the organizationand degree of hydrogen bonding in the glucan solution overall. Theskilled reader is aware of the impact on a glucan solution of, forexample, 150 mM NaOH and the concentration of other hydrogen bondbreakers can be selected accordingly. The purpose of the second step,where conditions are provided which allow reestablishment of hydrogenbonds, is effectively to rapidly neutralise or reverse the effect on thepotential for intermolecular electrostatic interactions caused by theaddition of the hydrogen bond breaker. Thus the nature and concentrationof this second agent will follow from the selection of the hydrogen bondbreaker. It is also important to mention that the conditions of rapidneutralization provide for the “freezing” of an energeticallymeta-stable supermolecular structure which, without rapid neutralizationwould otherwise tend to re-organize in an energetically more optimal,but less bioactive manner. Further processes for increasing thestability of the final product resulting from the treatment according tothe present invention, can of course be evaluated. Possible additionalmethods could be the addition of stabilizers or any method to establisha energetically more optimal molecular structure thus enhancing thestability of the final product without impairing its biological activityprofile to a large degree.

In an industrial process the steps will conveniently be performed in atank large enough to hold the entire batch of product.

The steps of hydrogen bond disruption and then restoration as describedabove may be repeated, e.g. once more.

Preferably, the method comprises a further step c) in which the ions(e.g. Na⁺ and Cl⁻) added during steps a and b above are removed, forinstance via filtration. Methods of filtration are well-known in theart, for instance the product could be diafiltered over a tangentialfilter against the required volume of purified water.

The concentration of glucan in aqueous solution prior to the disruptionof the hydrogen bonds is preferably 1.5-6%, more preferably 2 to 4%,most preferably about 2%. Preferably, the concentration of glucan in theglucan gel is about 2%, for instance 1.8% to 2.2%. Therefore, preferablythe concentration of glucan in aqueous solution prior to the disruptionof hydrogen bonds is also about 2%. The addition of agents in steps a)and b) of the above methods may increase the volume of the aqueoussolution and so decrease the concentration of glucan in the solution.Preferably however the volume of agents added in steps a) and b) doesnot change the volume of the solution significantly, such that theconcentration of glucan in the starting and end products is roughlyequal. Of course, the skilled man will appreciate that, if desired, ahigher concentration of glucan in the starting product can be used suchthat the addition of the agents in steps a) and b) leads to a precise,desired glucan concentration in the final product. The skilled man willbe able to calculate the appropriate glucan concentration in thestarting product and the appropriate volumes of agents to add in stepsa) and b) to achieve a desired glucan concentration in the resulting gelproduct.

The above disruption and restoration of hydrogen bonding may beperformed on any aqueous solution of glucan molecules; preferredglucans, including glucans with modified branching, are discussed aboveand the glucan solution will preferably be a yeast glucan solution. Thestarting material may be a gel, in which case step a) results in anon-gel solution and step b) reinduces a gelatinous state. The weightaverage molar mass (M_(W)) of the glucans in the starting solution ispreferably high, preferably, on a single chain basis, the weight averagemolar mass of glucans in solution is above 15,000, more preferably above20,000, most preferably above 25,000 g/mol. Suitable methods fordetermining these mass values are given above.

The methods of the present invention include methods in which a 1% to 4%aqueous solution of soluble beta-glucan is the starting material, towhich is added NaOH at a concentration of about 2M to a finalconcentration of about 150 mM, the solution then being stirred untilfully solubilised and clear. Gel form is re-established by adding anequimolar amount of HCl under stirring, which results in a gel withphysiological osmolarity and pH of about 7. The gel can be produced inany volume.

The gels of the present invention can also be produced by a lay personwhen reagents, i.e. the starting material and the agents used in step a)and step b) are provided as a kit. Thus, in a further aspect the presentinvention provides a kit comprising a sealed vessel containing anaqueous solution of glucan molecules, a second sealed vessel containingan agent able to dissociate the glucan's hydrogen bonds and a thirdsealed vessel containing an agent able to reform hydrogen bonds withinthe glucan. The aqueous solution of glucan molecules, and the two agentscomprising the kit may be as defined anywhere herein.

One such kit comprises a bottle of 85 ml 2.2% beta glucan in aqueoussolution, a sealed tube of 7.5 ml 2M NaOH and a sealed tube of 7.5 ml 2MHCl, where the two latter reagents are added successively to the bottleof 2.2% beta glucan giving an isotonic 2% final gel. A further exampleis a kit comprising a bottle containing 70 ml 4% beta glucan in aqueoussolution, a sealed tube of 15 ml 1M NaOH and a sealed tube of 15 ml 1MHCl, which results in 100 ml of a gel with concentration approximately(a little less than) 3% when the latter two reagents are addedsuccessively to the bottle of beta-glucan.

Glucans are generally extracted from their source material (e.g. fungi,yeast or cereal) in particulate form but methods of generating solubleforms from particulate glucans are known in the art and include acid oralkali treatments, such as the formolysis step described in WO 95/30022and for instance various types of glucans from cereals like barley fromSigma Chemical. According to the present invention, a particulatestarting material, such as may be prepared by the protocol in Example 1of WO 95/30022, will preferably be solubilised by heating in formic acidfor at least two hours. Formolysis performed on particulate glucanstarting material may conveniently cause selective removal of any β(1,6)linked glucosyl side chains as well as solubilising the particulateglucan.

The methods of the invention also optionally comprise a heating stepwhere the formic acid treated product is boiled (>100° C.) for at least30 mins. After the product has cooled it is preferably treated to removeparticulate materials by regular methods know in the art e.g. bycentrifugation or filtration.

The particulate glucan which is treated to yield a soluble form forprocessing in accordance with the present invention is preferablyderived from cell walls, in particular yeast cell walls, which have hadthe protein components and other remnants like mannan and chitin removedtherefrom e.g by washing.

One example of a suitable particulate yeast glucan product is producedby Biotec Pharmacon ASA which is derived from Bakers Yeast(Saccharomyces cerevisiae) and known as NBG COS®. Another example ofparticulate glucan raw materials are whole glucan particles like theproduct Imprime WGP™. The product is a natural underivatized (in termsof chemical modifying groups) particulate β(1,3)/(1 ,6) glucan,characterised by NMR and chemical analysis to consist of polymers ofbeta-1,3-linked D-glucose containing side-chains of beta-1,3 andbeta-1,6-linked D-glucose.

The visual appearance of preferred gel products of the present inventionis firm, opaque, whitish with a high adhesion capacity to othersurfaces.

In a further aspect the present invention provides a glucan productobtained or obtainable by any of the aforementioned processes.

The glucans of the present invention are potent therapeutic agents andin a further aspect the present invention provides the glucans asdescribed herein for use in therapy, in particular for the treatment ofconditions where a subject is in need of a systemic or local enhancementof the immune response, e.g. where there is tissue damage or infection.The glucans are of particular utility in assisting wound or ulcerhealing and in the treatment of oral mucositis and cancer or reducingtumour size.

In a further aspect the present invention provides therefore a method ofassisting wound or ulcer healing or treating oral mucositis in a subjectin need thereof which comprises administration to said subject of aglucan of the present invention as described herein.

Reference is made to “assisting” wound or ulcer healing because somewounds or ulcers will heal naturally and others may not but the glucansof the invention have been shown to accelerate wound and ulcer healing.In some cases, healing may not occur satisfactorily without treatment.An example for such a wound which demands treatment for healing isdiabetic foot ulcer. In this indication the patient develops woundsbased on the underlying cause which is diabetes. Due to the oftenuntreated underlying cause and the fact that these wounds are to befound on the feet of patients, these ulcers do not heal by themselvesand cause huge problems for the patient usually ending in amputation ofthe foot.

In a further aspect the present invention provides a method of treatingcancer or reducing the size of a tumour in a subject which comprisesadministration to said subject of a glucan of the present invention asdescribed herein. Preferably the glucan is administered orally.Preferably, the glucan is administered at a dosage of 5 to 200mg/kg/day, more preferably 20 to 100 mg/kg/day.

In a further aspect the present invention also provides a pharmaceuticalcomposition comprising a glucan in gel form as defined above and one ormore pharmaceutically acceptable diluents or carriers, preferably waterand optionally one or more physiologically acceptable stabilisers orfurther diluents or carriers. The compositions may conveniently beformulated into any topical dosage form. The topical dosage forms may begels, pastes, creams, sprays, lotions, solutions, ointments, films, etc.

In some variations, the compositions as described herein are in the formof an ointment. The ointment base may be an oleaginous base, anemulsifiable base, an emulsion base, or a water-soluble base. In othervariations, the compositions according to the present invention are inthe form of a cream. The creams may be viscous liquids or semisolidemulsions, either oil-in-water or water-in-oil. The cream bases may bewater-washable, and contain an oil phase, an emulsifier, and an aqueousphase. In yet further variations, the compositions of the presentinvention are in the form of a lotion. The lotions may be formulated assuspensions of solids and contain suspending agents to produce betterdispersions. The compositions according to the present invention mayalso be formulated pastes. Pastes are semisolid dosage forms in whichthe active agent is suspended in a suitable base. Depending on thenature of the base, pastes are divided between fatty pastes or thosemade from a single-phase aqueous gels.

In some variations, the compositions form a film on the wound surface.This film can be applied by a spray or other suitable means. To aid filmformation, film forming agents such as, but not limited to, acrylic acidand its derivatives, polyacrylic and its derivatives such aspolybutylmethacrylate and polymethacrylic acid, polymethacrylate,ascorbyl palmitate, carbomer, carnauba wax, cellulose derivatives suchas cellulose acetate phthalates, rosca mellose sodium, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, ethylcellulose and related compounds, hydroxypropyl methylcellulosephthalate, hypromellose phthalate, cetyl alcohol and derivatives,microcystalline wax, poloxamer, polyethylene glycol, polyurethane,polyvinyl acetate, polyvinyl acetate phthalate, polyvinyl alcohol,silicone rubber and derivatives, shellac, triglycerides derivatives, andcombinations thereof are used.

The compositions can also include at least one film plasticizer agentthat may serve to soften the polymer film formed by the film formingagent so that it is sufficiently flexible to move with area of the bodyapplied without cracking or peeling.

In some variations, the compositions may be cast into a film prior toapplication to the wound or applied to the wound directly where theypolymerize in situ. A “spread-on” film polymerizes when applied to theskin and may be delivered as a cream or ointment from a tube, roll-on,spray, and the like. The film may be created by incorporating a siliconerubber, into the external phase. Upon mixing with the internal phase,the resultant emulsion is allowed to cure and provides a “spread-on”film, which polymerizes when applied to the wound. The emulsion may bespread onto a substrate to achieve a desired thickness.

In other instances, the compositions may be preformed into a layer orpatch. The patch may be of varying thickness. The patch may also be cutto have a shape that generally follows the wound edges.

In some variations, the patches may include a pharmaceuticallyacceptable adhesive material that serves to affix the patch to the woundor skin. A patch backing layer may also be included.

The compositions may be directly placed on a wound, or placed on asubstrate for application on a wound. Any substrate (carrier) may beused with compositions described here. For example, woven, non-woven,knitted, foam, and adhesive substrates may be used. Absorbent ornon-absorbent substrates may also be used. In some variations, thecompositions are sprinkled or spread on the substrate. In othervariations, the compositions are impregnated within the substrate.

The wound dressings may be applied for any suitable time period. Forexample, they may be applied over a time period of one day, over severaldays, over several weeks, or for several months or more. In general, thewound dressings will be reapplied until the wound is healed. Theduration of wound treatment with the dressings described here may dependon such factors as the type of wound being treated, wound location,wound exudates, and form of the composition being applied. Depending onthe form used, the composition may be removed with water, or wiped orpeeled off the wound.

The compositions described here may be used to treat wounds resultingfrom any etiology. For example, the wounds may be due to burns,infections, ischemia, lymphedema, neoplasms, neuropathy, radiationdamage, surgical procedures, venous insufficiency, and trauma. Thecompositions of the present invention are of particular utility inassisting wound or ulcer healing.

The invention further provides a physical support, for example anymedical device or material for medical use having applied thereto,including impregnated therein, a glucan of the invention as definedherein.

One important characteristic of such beta glucans is their water holdingcapacity and gel formation characteristics even in the absence ofconditions like non-neutral pH or cations which might promote gelhealing. Some beta-glucans would form gels at concentrations as low as1%, but more typically in the range of 2-4%. A soluble beta-glucan fromyeast like the one described herein will form a thixotropic andpseudoplastic gel when dissolved in aqueous solution at a concentrationof 1-6% in pH range from 3-7, independent of the presence of cations.

The compositions of the invention comprise 1.5-6%, preferably 1.5-5%beta glucan in an aqueous solution, preferably the composition comprisesaround 2-3% glucan in an aqueous solution. The use of differentconcentrations is dependent on the purpose and the different modes ofadministration. As a general rule, a yeast glucan with a concentrationof more than 6% in an aqueous solution and free from other stabilizingsubstances would result in a final gel product which is difficult tomanufacture due to its solid gel properties.

Encompassed by the terms ‘wound’ and ‘ulcer’ are surface wounds,surgical wounds, burns, open fractures, leg ulcers, apthous ulcers,diabetic ulcers and decubitus ulcers. Wounds may be as a result ofinjury, surgery or disease but all are characterised by a loss of dermalintegrity, the skin may be torn, cut or punctured and regrowth of theskin is required to seal the opening. The glucans of the presentinvention have been shown to accelerate wound closure. As shown in theExamples, efficacy can readily be demonstrated by measuring the size ofan open wound.

The compositions are preferably applied topically, e.g. as a gel,transdermal patch, lotion, ointment, cream etc. Compositions may beapplied daily, more frequently or less frequently, e.g. twice daily oron alternate days and for a duration as determined by a clinician or insome cases by the patient or other health advisor. The duration oftreatment will depend on the nature and severity of the wound or ulcerwith progress generally being readily determined by visual inspection.

Topical administration includes administration in the mouth and suitablegels, pastes, sprays, lozenges, etc. for delivery to the oral mucosa areknown in the art.

The glucans and compositions containing them find utility in human andveterinary medicine. As used herein, the term ‘medical’ includesveterinary applications and contexts. Humans are preferred subjects fortreatment but other animals which may usefully be treated includelivestock and companion animals.

The glucans of the invention and compositions containing them may beapplied to or incorporated in a physical/solid support such as a patch,dressing, plaster, bandage, film, gauze etc. which can be applied to thewound or ulcer site and such products constitute a further aspect of thepresent invention.

The glucans of the present invention also find corresponding utility inin vitro applications for the culturing of skin cell lines, e.g. for usein skin grafts. Thus in a further aspect the present invention providesan in vitro method of proliferation of skin cells which comprisescontacting a population of skin cells with glucans of the invention asdescribed herein.

It will be appreciated that preferred features applicable to one aspector embodiment of the invention apply, mutatis mutandis, to all aspectsand embodiments.

The glucans of the present invention have excellent in vivo efficacy aswound healing and anti-cancer agents, as shown in the Examples. TheExamples also show the ability of the glucans of the invention tostimulate production of cytokines which are relevant in a variety oftherapeutic contexts. The Examples show that the glucan of the presentinvention has different biological activity, as demonstrated byinduction of cytokine production, as compared to a superficially similarglucan product which is also obtained from yeast, is soluble and hasbeen treated to selectively reduce the (1,6) linked side chains whileretaining (1,3) linked side chains. In particular, the glucan of thepresent invention can induce the differentiation of human myeloiddendritic cells towards an inflammatory phenotype, significantlystimulate TNF-alpha secretion and induce expression of G-CSF and IL-10by these cells, while the secretion of CXCL-10 is basically at baselinelevel, and appears to be unaffected by the treatment described herein.This is important and illustrates that the preferred glucan of thepresent invention stimulates the secretion of a specific set orcombination of cytokines. The glucan of the present invention can alsostimulate macrophages from diabetic mice (db/db) to secrete CXCL2, PGE2and GM-CSF, which all have prominent roles in wound healing. Inaddition, the gel glucans of the present invention activate the humancomplement system. The effect of the preferred beta glucans on releaseof TNFα is dose-dependent and appears to diminish at glucanconcentrations above a certain threshold value eg. 100 μg/ml in avariant of the RAW cell line overexpressing the beta glucan receptordectin-1. Both the concentration yielding the maximal TNFα secretion,and also the magnitude of the response is higher compared to what isseen using a soluble beta glucan that has not been subjected to thetreatment described herein.

The invention will now be further described in the followingnon-limiting Examples and the figures in which:

FIG. 1 shows storage modulus, G′ (Pa), plotted against temperature for aglucan gel according to the present invention. The data was obtained bysmall strain oscillatory measurements using a Stresstech HR rheometerand the following temperature scan: 70 to 10° C. at a rate of ⅓° C./min,kept at 10° C. for 2 h and then 10 to 70° C. at a rate of ⅓° C./min. Themelting temperature of this gel (gel to sol) is determined toapproximately 40 ° C. based on where the increasing temperature curvelevels out (G′≈0 Pa).

FIGS. 2 a) to e) show in vitro stimulation of human mDC with differentbatches and concentrations of SG (081-5, 252-7, 342-8, 421-4) anddifferent concentrations of SG-LS (421-4 new). The concentration ofsecreted a) TNFα, b) G-CSF, c) IL-10, d) CXCL-10 and e) IL-12p70 areindicated along the y-axis.

FIGS. 3 a) to e) show in vitro costimulation of human mDC with LPS inand different batches and concentrations of SG (081-5, 252-7, 342-8,421-4) and different concentrations of SG-LS (421-4 new). Theconcentration of secreted a) TNFα, b) G-CSF, c) IL-10, d) CXCL-10 and e)IL-12p70 are indicated along the y-axis.

FIG. 4 shows secretion of CXCL2 by macrophages from db/db micestimulated by different concentrations SG and SG-LS. LS denote SG-LS.*p<0,05.

FIG. 5 shows secretion of PGE2 by macrophages from db/db mice stimulatedby different concentrations SG and SG-LS. LS denote SG-LS.

FIG. 6 shows secretion of GM-CSF by macrophages from db/db micestimulated by different concentrations SG and SG-LS. LS denote SG-LS.

FIG. 7 shows the secretion of TNFα by a dectin-1 over-expressing RAWcell line stimulated by either SG (421-4), SG-LS (421-4 LS). Phospahatebuffered saline served as a negative control.

FIG. 8 shows SG 131-9 2% and potentiated (L/S) 131-9 2% versus vehicle(water) and positive control (rh-PDGF-BB (10 μg)+rh-TGF-α (1 μg) in 0.5%HPMC), mean ±s.e.m. *p<0.05. **p<0.01.

FIG. 9 shows SEC-MALS-RI chromatograms in aqueous solution of apotentiated glucan produced according to the present invention togetherwith the starting glucan prior to treatment with alkali and acid. Theprofiles are similar, but it is apparent that very high molecular weightaggregates have been removed by the procedure.

FIG. 10 shows growth of xenogeneic BT474 tumor cells transplantedintradermally into nude mice. SG (09BP003), SG-LS (09BP003) or water(solvent control) was given p.o. every second day from day 7 until day38.

FIG. 11 shows fluorescence in human in-vitro differentiated, bloodmonocyte derived, myeloid dendritic cells feed DTAF-stained SG (100μg/ml) and SG-LS (20 μg/ml) for 2h. DTAF was detected by FACS. Level ofDTAF was studied in immature cells pretreated by a dectin-1 bindingantibody (antagonist) (a), or mature mDC (b). The DTAF level inglucan-feed immature cells pretreated with PBS alone (a, b), served ascontrols (100%). Y-axis denotes percentage fluorescence compared tocontrols.

FIG. 12 shows fluorescence in human in-vitro differentiated, bloodmonocyte derived, myeloid dendritic cells feed DTAF-stained SG (100μg/ml), SG-LS (20 μg/ml), dextran and luciferase yellow (LY) for 2 h.Dextran is a clathrin-dependent control ligand, while LY is afluid-phase (macropinocytosis) marker. DTAF was detected by FACS. Levelof DTAF was studied in immature cells pretreated by the indicatedinhibitors. The DTAF level in glucan-feed immature cells pretreated withPBS alone served as controls (100%). Y-axis denotes percentagefluorescence compared to controls.

EXAMPLES Example 1

Preparation of Gel Glucan Product of the Present Invention (SG-LS)

An aqueous solution of 2% yeast glucan molecules was treated asdescribed below. This aqueous solution was prepared from a particulateglucan preparation by formolysis to selectively remove β-1,6 side chainsand subsequent purification and diafiltration to remove particulatematter and low molecular weight components from the formolysis solution.A suitable formolysis step is disclosed in Example 3 of EP 0759089 B1.The particulate glucan was itself prepared from cell walls of Baker'sYeast (S. cerevisiae) by separate extractions with alkali, ethanol andwater, each extraction being followed by appropriate drying (spraydrying and vacuum drying).

a. Disruption of Hydrogen Bonds By Addition of Sodium Hydroxide:

Addition of sodium hydroxide took place after the concentration of theglucan solution had been adjusted, giving a product volume ofapproximately 200 litres in a closed and agitated 800 litre tank whichis heated or cooled by introduction of steam or water to a jacketsurrounding the tank.

The product temperature was adjusted to 18° C., and 24 moles of NaOH,dissolved in approximately 10 litres of purified water, was pouredslowly (approximately 1 litre per minute) through a hatch in the tank.

b. Restoration of Hydrogen Bonds By Addition of Hydrochloric Acid:

The restoration process was started immediately after the last of theNaOH has been poured into the tank.

Slightly less than 24 moles of HCl, approximately 9 litres of a 2.4Msolution in purified water, was poured into the tank relatively quickly(in approximately 2 minutes), the pH of the product was measured, andmore acid added in small portions until pH reached approximately 4.

c. Removal of Salt

To remove the ions (Na⁺ and Cl⁻) added during steps a and b, the productcan be diafiltered over a tangential filter against the required volumeof purified water.

Example 2

Stimulation of Human Dendritic Cell Maturation

The potency of different formulations of soluble beta-glucan todifferentiate monocyte derived immature dendritic cells (iDC) intomature dendritic cells (mDC) differs. The level of activation can bevisualised by measuring the expression of selected DC cell surfacemarkers.

Human monocytes purified by lymphoprep gradient followed by magneticcell sorting (MACS) with anti-CD14 microbeads were cultured for 5 dayswith a combination of IL-4 and recombinant human GM-CSF to promote thedifferentiation into immature dendritic cells. The monocyte derivedimmature dendritic cells (iDC) were cultivated at physO₂ levels. Fromday 5 to day 6 the iDC were stimulated with 50 μg/ml soluble beta-glucan(SG), or 10 μg/ml non-soluble beta glucan (NG). Expression of thesurface molecules HLA-DR, CD83 and CD86 were used to survey thedifferentiation of iDC into mature DC, and were analyzed by fluorescentactivated cell sorting FACS. Also expression of C-type lectin receptorDC-SIGN was analysed.

Compared to the negative control (PBS) soluble glucan (SG), which is thepost-formolysis, pre-NaOH treated glucan of Example 1 and is a glucanpresent in aqueous solution at a concentration of 2%, slightlydownregulates the expression of CD83, CD86, MHC class II (HLA DR) andDC-SIGN. The down regulation is primarily a result of a lover number ofcells expressing the protein, while expression of the CD86 protein isslightly down regulated per cell as well. In contrast, SG-LS, a glucanaccording to the present invention and prepared in accordance withExample 1, is a powerful stimulus which activates iDCs to upregulate theexpression of CD83, CD86, and HLA-DR. Also in contrast to SG theexpression of DC-SIGN is efficiently down regulated by SG-LS.Non-soluble beta glucan from Saccharomyces cerevisiae activates asimilar pattern of protein expression of CD83, CD86, HLA-DR and DC-SIGNas SG-LS, although even more powerful. Down regulation of DC-SIGN inconjunction with up regulation of CD83, CD86 and MHC class II areaccepted hallmarks of dendritic cell activation. Thus, SG-LS activatesdendritic cells in vitro, while SG does not, and that the properties ofSG-LS with respect to this function resembles non-soluble beta glucanparticles from S. cerevisiae.

Example 3

Stimulation of Cytokine Secretion by Human Dendritic Cells (DCs)

To determine the cytokine profile secreted by human DCs in vitro,peripheral blood monocytes were isolated and propagated into mDC usingstandard methods. The mDCs were subsequently stimulated with differentconcentrations of soluble beta-glucans, either alone or in concert withbacterial lipopolysaccharide (LPS) (1 ngml⁻¹). The cytokine profile wasdetermined by multiplex analysis using the Luminex system. FIG. 2 showsthat SG stimulation leads to a weak induction of TNFα secretion, whereasG-CSF, IL-10, CXCL-10 and IL-12 remain unaffected. In contrast, SG-LS(“421-4 new” in FIG. 2) strongly stimulates TNFα secretion as well as alow level secretion of both G-CSF and IL-10.

SG is not a glucan in accordance with the invention, but can bepotentiated accoding to the presented protocol as illustrated by SG-LS,which is a gel glucan product in accordance with the present inventionand prepared in accordance with Example 1.

The secretion of CXCL-10 was, as for SG, not affected by SG-LSstimulation, while the production of IL-12 was weakly inhibited bySG-LS.

Costimulation of human mDCs with SG or SG-LS together with LPS revealedthat SG-LS has a synergistic or additive effect on the secretion ofTNFα, CXCL-10, IL-10, and G-CSF, while secreation of IL-12 was clearlydownregulated compared to LPS alone (FIG. 3). Costimulation of SG andLPS did not induce any clear changes in any of the cytokines tested(FIG. 3).

Taken together, SG and SG-LS induce distinctive biological functionsfrom in vitro stimulated human mDC.

The example shows that the soluble glucan produced according to thepresent invention has a stronger ability to modulate the effect of otherpathogen associated molecular patterns as compared to a soluble glucannot subjected to the procedure described herein.

Example 4

Stimulation of Cytokine Secretion by Mouse Macrophages

Macrophages from diabetic (db/db) mice (BKS.Cg-mDock7^(m)+/+Lepr^(db)/J) were harvested by intraperitoneal lavage usingPBS supplemented by EDTA. The cells were seeded in microplates andstimulated with either SG or SG-LS for 12 h at 37° C., either alone orin combination with LPS. The supernatant was analyzed by ELISA for aseries of signaling molecules involved in wound healing andinflammation.

Both SG and SG-LS stimulated macrophages from the db/db mouse to secreteCXCL2 (FIG. 4, The concentration of the secreted chemokine in thesupernatant from the SG stimulated cells were not significantlydifferent from the what was measured from cells given phosphate bufferedsaline only. In contrast, cells given SG-LS secreted significantly moreCXCL2 than the control cells.

Macrophages from db/db mice stimulate by SG-LS secrete PGE2 (FIG. 5) andGM-CSF (FIG. 6). Due to a high variation in the assay the concentrationsin the supernatants of either signaling molecule were not significantlydifferent from the concentrations in the supernatants from cellsincubated in phosphate buffered saline. On the other hand, SG did notstimulate secretion of either PGE2 or GM-CSF (FIGS. 5 and 6,respectively).

Example 5

Stimulation of TNFα Secretion by RAW/Dectin-1 Cell Line

The RAW/dectin-1 cell line is a stable transfectant of the RAW264.7mouse leukaemic monocyte macrophage cell line over-expressing thebeta-glucan receptor, dectin-1. The cell line corresponds to the RAWblue™ cell line from Invivogen. The cell line is suitable to determineindividual differences between different formulations of soluble betaglucan, and the beta glucan response mounted by this cell line isindicative of an interaction with the dectin-1 receptor. Both SG andSG-LS induce secretion of TNFα as measured in an ELISA based assay 24 hafter stimulation at 37° C. (FIG. 7). Both formulations induce a typicaldose-response. The maximal effect of SG is approached by 1-2 μg/ml, anddeclines at lower or higher concentrations. In comparison the maximaleffect of SG-LS is seen at 100 μg/ml giving rise to a 5-fold higherconcentration of TNFα in the medium surrounding the cells.

Thus, both SG and SG-LS stimulates a dectin-1 over-expressing murinecell line to serete TNFα, but the responses are characteristic andeasily distinguishable. While the response to SG diminish above 4 μg/ml,the response to SG-LS becomes stronger until 100 μg/ml. This suggeststhat SG and SG-LS interacts differently to the major beta glucanreceptor, dectin-1.

Example 6

Wound Healing In Vitro

The impact of SG and SG-LS, respectively, on wound healing wasinvestigated by analysing the repair of full-thickness excisional skinwounds in the diabetic (db/db) mouse model (i.e. BKS.Cg-mDock7^(m)+/+Lepr^(db)/J mice). Upon acclimatisation (5-7 days withoutdisturbance) the animals were housed in groups of 5 animals according toHome Office regulations and the specific requirements of diabeticanimals. After experimental wounding, animals were housed in individualcages (cage dimensions 35×15×15 cm with sawdust bedding, changed twiceweekly), in an environment maintained at an ambient temperature of 23°C. with 12-hour light/dark cycles. The mice were provided with food(Standard Rodent Diet) and water ad libitum. Following all anaestheticevents, animals were placed in a warm environment and monitored untilthey were fully recovered from the procedure. All animals receivedappropriate analgesia (buprenorphine) after surgery and additionalanalgesics as required. All animal procedures were carried out in a HomeOffice licensed establishment under Home Office Licences (PCD: 50/2505;PPL: 40/3300; PIL: 50/3482; PIL: 70/4934). The health of animals was illmonitored on a daily basis throughout the study.

On day 0, animals were anaesthetised (isofluorane & air) and the dorsumshaved and cleaned with saline-soaked gauze. A single standardisedfull-thickness wound (10.0mm×10.0mm) was created in the left dorsalflank skin of each experimental animal. Wounds in all treatment groupswere subsequently dressed with a circumferential band of the transparentfilm dressing Bioclusive™ (Systagenix Wound Management, UK); after whichthey received either SG or SG-LS by injection 50 μl of a 2% solution inpurified water through the Bioclusive film using a 29-gauge needle.Diabetic animals were randomized to one of the treatment regimes usingappropriate software. For the experimental groups receiving either SG orSG-LS treatments was reapplied on post-wounding days 2, 4 and 6. Woundsites in these animals were closely monitored for excessive build-up ofapplied agents and excessive wound site hydration; if excessive appliedagent accumulation/hydration was apparent, previously applied materialwas removed by aspiration prior to reapplication. For the positivecontrol group treatments was reapplied daily until post-wounding day6—wounds in this group received a total of 7 applications of the growthfactor combination treatment. On post-wounding days 4, 8 and 12 allanimals were re-anaesthetised, their film dressings and any free debrisremoved, and their wounds cleaned using saline-soaked sterile gauze.After photography on days 4 and 8, wounds were re-dressed as above withBioclusive film dressing.

Wound closure data were determined from scaled wound images taken ofeach wound at each assessment point. The area of a given wound, at agiven time point, was expressed as a percentage of the area of thatwound immediately after injury (i.e. day 0). The mean percentage woundarea remaining (& standard error of mean) was calculated for each groupand was displayed graphically (FIG. 8). The impact of each glucanpreparation was compared to that of wounds in receipt of: i). vehicle(water); and ii) PDGF-BB+TGF-α (positive control).

Wounds in receipt of SG 131-9 LS 2% displayed elevated wound closure,relative to wounds in receipt of SG 131-9 2%, at all time pointsassessed (FIG. 8). This observed difference was statisticallysignificant at days 4 and 8 (p=0.015 & 0.001 respectively). At the earlytime points (days 4 and 8) the wound closure profile of the SG 131-9 LStreated wounds was comparable to that of positive controltreated-wounds.

Example 7

Determination of Melting Point

Determination of the melting point of a glucan gel produced according tothe present invention was performed as described in the description andthe results are shown in FIG. 1. The alkali-acid treatment generallyincreases the melting temperature (gel to sol) of the glucan gel.

Example 8

The impact of SG and SG-LS, respectively, on anti-tumour activity wasinvestigated using NMRI nu/nu mice with an intradermal transplant of 10⁷BT474 cells in matrigel. After a period of tumour growth, 7 days untilpalpable (80 mm³), SG and SG-LS were administrated daily by oral cavage.Tumour diameter was measured every second day over a 31-day period, andvolumes determined. The analysis (FIG. 10) revealed that both SG andSG-LS delayed tumour growth compared to the vehicle (water). It was alsoclear that SG-LS inhibited the growth rate more efficiently thancompared to SG, suggesting that also the anti-cancer properties of SGare potentiated by the herein described method of production.

Example 9

The difference in efficacy between SG and SG-LS was investigated byanalysing their mechanisms of cellular interaction and the results areshown in FIGS. 11 and 12. Uptake of the LS variant in human in-vitrogenerated myeloid dendritic cells derived from blood monocytes (mDC) isinhibited by a dectin-1 antagonist (anti-dectin-1 antibody, FIG. 11a ).Uptake of SG was only slightly inhibited by the antibody suggesting thatSG enters the cell primarily by mechanism independent of dectin-1. Thisfinding was further substantiated by studying the uptake offluorescein-labeled glucans in mature mDC and immature mDC. It is wellknown that the surface expression of dectin-1 is lower in maturerelative to immature mDC, and hence uptake of SG-LS in mature mDC isreduced (˜30%, FIG. 11b )) compared to immature mDC (100%, not shown).The uptake of SG was similar in both mature and immature mDC, supportingthe dectin-1-antagonist data, and suggesting that SG-LS and SG interactsdifferently with the cells.

The precise mechanisms were determined using specific inhibitors (FIG.12). Uptake of SG was unaffected by chlorpromazine, while endocytosis ofSG-LS was inhibited by this compound. This suggests that SG-LS is takenup by clathrin-mediated endocytosis, while SG is not. On the other hand,uptake of SG is inhibited by rottlerin which interferes withmacropinocytosis. Intracellular accumulation of SG-LS is not affected byrottlerin. Cytochalasin D partially inhibits the uptake of both ligands,suggesting a requirement for cytoskeleton rearrangements, i.e.phagocytosis, to enter the cells. Taken together these resultsdemonstrate that SG and SG-LS are taken up by a different mechanisms,although phagosytosis is common to both.

The invention claimed is:
 1. A gel glucan product comprising a solubleyeast glucan in aqueous solution at a concentration of 1 to 6%, theglucan having a weight average molar mass of 15,000 to 50,000 g/mol on asingle chain basis and a weight average molar mass in aqueous solutionon an aggregate basis of 4 to 20×10⁵ g/mol, the gel glucan producthaving a gel to sol melting temperature of 35 to 60° C.
 2. The gelglucan product of claim 1, wherein the glucan has a weight average molarmass of 20,000 to 40,000 g/mol on a single chain basis.
 3. The gelglucan product of claim 1, wherein the glucan has a melting temperature(gel to sol) of about 40° C.
 4. The gel glucan product of claim 1,wherein the glucan is in aqueous solution at a concentration of about2%.
 5. The gel glucan product of claim 1, wherein the glucan is derivedfrom Saccharomyces cerevisiae.
 6. The gel glucan product of claim 1,wherein the glucan is a beta glucan comprising a backbone ofβ-(1,3)-linked glucosyl residues and side chains comprising 2 or moreβ-(1,3)-linked glucosyl residues, the sidechains being attached to thebackbone via a β-(1,6)-linkage.
 7. The gel glucan product of claim 1,wherein the glucan is essentially free of repetitive β-(1,6)-linkedglucosyl residues.
 8. A method of producing a gel glucan product, saidmethod comprising the following steps: a) treating an aqueous solutionof soluble glucan molecules with an agent which dissociates the glucan'shydrogen bonds; and b) contacting the product of step a) with an agentwhich enables reformation of hydrogen bonds within the glucan; whereinin step a) the glucan is derived from yeast and is in aqueous solutionat a concentration of about 1% to wbout 6%, the glucan having a weightaverage molar mass of 15,000 to 50,000 g/mol on a single chain basis anda weight average molar mass in aqueous solution on an aggregate basis of4 to 20×10⁵ g/mol.
 9. The method of claim 8, wherein the agent whichdissociates the hydrogen bonds is an alkali salt, sodium hydride, sodiumamide, urea or formamide.
 10. The method of claim 9, wherein the agentwhich dissociates the hydrogen bonds is sodium hydroxide.
 11. The methodof claim 8, wherein the agent which dissociates the hydrogen bonds isused at a final concentration of above 50 mM.
 12. The method of claim11, wherein the agent which dissociates the hydrogen bonds is used at afinal concentration of about 150 mM.
 13. The method of claim 8, whereinthe agent which enables reformation of hydrogen bonds is a strong acid.14. The method of claim 13, wherein the acid is hydrochloric acid,formic acid, sulphuric acid, phosphoric acid, acetic acid or citricacid.
 15. The method of claim 8 wherein step b) results in gel formationin less than 10, preferably less than 4 minutes.
 16. The method of claim8, wherein the agents of step a) and b) are added in equimolar amounts.17. The method of claim 8 which is preceded by a formolysis step whereina particulate glucan starting material is suspended in formic acid inorder to remove β-(1,6) linked glucosyl side chains and to solubilisethe particulate glucan.
 18. A gel glucan product obtainable by themethod of claim
 8. 19. A pharmaceutical composition comprising the gelglucan product of claim 1 and one or more pharmaceutically acceptablediluents or carriers.
 20. A pharmaceutical composition comprising thegel glucan product of claim 18 and one or more pharmaceuticallyacceptable diluents or carriers.